Desintegrin-like and cystein-rich domains isolation from a Bothrops alternatus hemorrhagin.

GAY CLAUDIA C.; LEIVA LAURA C.; ACOSTA, OFELIA C.; BISCOGLIO DE JIMÉNEZ BONINO, MIRTHA

Rosario

Congreso; Sociedad Argentina de Investigación en Bioquímica y Biología Molecular. 42TH Annual Meeting.; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Snake Venom Metalloproteinases (SVMPs) are multiple domain enzymes whose principal toxic effects are due to disruption of the hemostatic system. That from Bothrops alternatus herein studied belongs to the PIII class (SAIB 2005). These enzymes can undergo autolysis to yield a biologically active product, comprising the disintegrin-like and the cystein-rich domains (DC domain). In this work we optimized the hemorrhagin isolation procedure by means of a combination of gel filtration and ion-exchange chromatography. The purified protein was submitted to autolysis ?in vitro? and SDS-PAGE showed fragments in the 28-45 kDa range. The 28 kDa band was electroblotted onto a PVDF membrane and - when submitted to Edman degradation - proved to be the metalloproteinase DC domain on the basis of the comparison of its twenty residue N-terminal sequence with those of Bothrops disintegrins included in data bases. On the other hand, the mixture from the autolytic process was resolved through a Superdex G75 column and fractions analyzed by ESI HPLC-MS. Different experimental conditions - such as EDTA incubation - were utilized for the purpose of stabilizing the enzyme in order to perform structural studies.